The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis

Abstract

The intrinsic damage response is activated by DNA damage that arises during the cell division process. The ability of the cell to repair this damage during proliferation is important for normal cell growth and, when disrupted, may lead to increased mutatagenesis and tumorigenesis. The atypical CDK activator, Spy1, was previously shown to promote cell survival, prevent apoptosis and inhibit checkpoint activation in response to DNA damage. Prior studies have shown that Spy1 is upregulated in breast carcinomas and accelerates mammary tumorigenesis in vivo. In this report, first, we demonstrate that the ability of Spy1 to inhibit apoptosis and bypass UV-induced checkpoint activation is dependent on the presence of the gene regulatory protein p53 and the CKI p21. Second, we demonstrate that Spy1 expression has the following effects: prevents repair of cyclobutane pyrimidine dimers through bypass of nucleotide excision repair; increases the cellular mutation frequency; and reduces the formation of cyclin E induced γH2A.X foci. Lastly, we show that knockdown of endogenous Spy1 leads to γH2A.X foci formation, Chk1 phosphorylation and proliferation defects, demonstrating a functional role for Spy1 in the intrinsic DNA damage response. These results also demonstrate that Spy1 fulfills a novel regulatory role in the intrinsic DNA damage response and maintains the balance between checkpoint activation, apoptosis, repair and cell cycle progression in response to exogenous or intrinsic damage. Furthermore, the overexpression of Spy1 as a contributing factor in cancer progression will most likely be confined to p53-positive cells.

The Atypical Cyclin-Like Protein Spy1 Overrides p53-Mediated Tumour Suppression and Promotes Susceptibility to Breast Tumorigenesis

“…Cells from MMTV-Spy1 mice had significantly more γH2AX positive cells at 24 hours post UV as compared to control littermate cells ( Figure 5E). Data from the MMTV-Spy1 mouse both in vivo and in vitro shows a significant increase in γH2AX following DNA damage, which is in opposition to previously published data, which shows a significant decrease in γH2AX with Spy1 overexpression (13,16). To determine if this is due to a difference in the time points studied, HC11 cells were transfected with pCS3 or Myc-Spy1-pCS3, UV irradiated and studied at a wide time course.…”

“…Interestingly, with only a modest decrease in p53 protein levels ( Figure 3A; middle panel) there was a very significant increase in Spy1 protein levels ( Figure 3A; left panel). Given that tumour formation was seen in a cell line with non-functional p53, and Spy1 can prevent checkpoint activation (13,15,16,20), it is plausible then that wild-type p53 may work to downregulate Spy1 to allow for p53 mediated cell cycle arrest, and elevated Spy1 with loss of p53 function would allow for enhanced genomic instability. To test the ability of wild-type p53 to regulate levels of Spy1 protein, mammary cells with mutated p53 (HC11 and MDA-MB-231 cells) were transfected with pEIZ, pEIZ-Spy1, p53 or pEIZ-Spy1 and p53 and lysates collected at 24 hours for western blot analysis.…”

“…An atypical cyclin-like protein Spy1 (also called Ringo, Speedy1; gene SPDYA) was initially discovered in a screen for genes that would override cell death following ultraviolet (UV) radiation in a rad1 deficient strain of S.pombe, suggesting a role for this protein in overriding critical checkpoint responses following DNA damage (12). Several groups have demonstrated that Spy1 is capable of inhibiting apoptosis and promoting progression through both G1/S and G2/M phase of the cell cycle (13)(14)(15)(16). Spy1 function is currently attributed to the direct binding to the cyclin dependent kinases (Cdks), activating both Cdk1 and Cdk2 independent of threonine 161/160 phosphorylation status (14)(15)(16)(17)(18)(19).…”

Identification of Male Gametogenesis Expressed Genes from the Scallop Nodipecten subnodosus by Suppressive Subtraction Hybridization and Pyrosequencing

“…This gene, also known as speedy , was first implicated as an initiator of oocyte maturation by inducing the G2/M transition in Xenopus oocytes (Ferby et al 1999, cited by [82]). It was later found to also induce G1/S phase progression when overexpressed in different human tissues, and more recently has been associated with promotion of tumors, possibly because of its capacity to prevent activation of checkpoints [83]. The fact that we did not detect any expression in maturing testis might be the result of this gene being under-expressed and not detectable by qPCR even if the subtraction was successful in isolating two identical reads that comprise the p33-ringo isotig.…”

Structural basis of divergent cyclin‐dependent kinase activation by Spy1/RINGO proteins

“…All Spy1 paralogs contain a highly conserved domain of about 130 amino acids called the Speedy/Ringo box (S/R box), which is required for Cdk binding and activation (Cheng et al, 2005b;Dinarina et al, 2005). Spy1 can directly bind and activate both Cdk1 and Cdk2, and Spy1 overexpression induces cells to progress faster through the cell cycle and to override checkpoints (Barnes et al, 2003;Gastwirt et al, 2006;McAndrew et al, 2009).…”